Cholic acid derivatives as 1,2,4,5-tetraoxane carriers: Structure and antimalarial and antiproliferative activity

Article abstract of DOI:10.1021/jm000952f

Cholic acid-derived 1,2,4,5-tetraoxanes were synthesized in order to explore the influence of steroid carrier on its antimalarial and antiproliferative activity in vitro. Starting with chiral ketones, cis and trans series of diastereomeric tetraoxanes were obtained, and the cis series was found to be ~2 times as active as the trans against Plasmodium falciparum D6 and W2 clones. The same tendency was observed against human melanoma (Fem-X) and human cervix carcinoma (HeLa) cell lines. The amide C(24) termini, for the first time introduced into the carrier molecule of a tetraoxane pharmacophore, significantly enhanced both antimalarial and antiproliferative activity, as compared to the corresponding methyl esters, with cis-bis(N-propylamide) being most efficient against the chloroquine-susceptible D6 clone (IC₅₀ = 9.29 nM). cis- and trans-bis(N-propylamides) were also screened against PBMC, and PHA-stimulated PBMC, showing a cytotoxicity/antimalarial potency ratio of 1/10 000.

Full text of DOI:10.1021/jm000952f

3274
J . Med. Chem. 2000, 43, 3274-3282
Ch olic Acid Der iva tives a s 1,2,4,5-Tetr a oxa n e Ca r r ier s: Str u ctu r e a n d
An tim a la r ia l a n d An tip r olifer a tive Activity1,†
Dejan Opsenica,# Gabriela Pocsfalvi,3 Zorica J uranic´, Bernard Tinant,§ J ean-Paul Declercq,§ Dennis E. Kyle,|
Wilbur K. Milhous,*,| and Bogdan A. Sˇolaja*,‡
Institute of Chemistry, Technology and Metallurgy, Belgrade, Yugoslavia, Centro Internazionale di Servizi di Spettrometria di
Massa del CNR di Napoli, Naples, Italy, National Cancer Research Institute, Belgrade, Yugoslavia,
Laboratoire de Chimie Physique et de Cristallographie, Universite´ Catholique de Louvain, Louvain-la-Neuve, Belgium,
Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100,
and Faculty of Chemistry, University of Belgrade, 11001 Belgrade, Yugoslavia
Received March 24, 2000
Cholic acid-derived 1,2,4,5-tetraoxanes were synthesized in order to explore the influence of
steroid carrier on its antimalarial and antiproliferative activity in vitro. Starting with chiral
ketones, cis and trans series of diastereomeric tetraoxanes were obtained, and the cis series
was found to be ∼2 times as active as the trans against Plasmodium falciparum D6 and W2
clones. The same tendency was observed against human melanoma (Fem-X) and human cervix
carcinoma (HeLa) cell lines. The amide C(24) termini, for the first time introduced into the
carrier molecule of a tetraoxane pharmacophore, significantly enhanced both antimalarial and
antiproliferative activity, as compared to the corresponding methyl esters, with cis-bis(N-
propylamide) being most efficient against the chloroquine-susceptible D6 clone (IC50 ) 9.29
nM). cis- and trans-bis(N-propylamides) were also screened against PBMC, and PHA-stimulated
PBMC, showing a cytotoxicity/antimalarial potency ratio of 1/10 000.
In tr od u ction
In continuation of our investigation on the synthesis
of steroidal tetraoxanes and the evaluation of their
antimalarial activity against P. falciparum African D6
and Indochina W2 clones,8a we prepared several cholic
acid-derived tetraoxanes differing in the C(24) substitu-
ent. The idea was to synthesize tetraoxanes with
protected polar groups (easily to hydrolyze in vivo)8b
thus possibly rendering solubility under physiological
conditions, to screen the activity of the compounds
derived from the natural carrier differing in solubility
within the series, and to investigate the influence of
steric effect of two predicted diastereomeric series on
their activity. In addition, the extensive in vitro anti-
proliferative activity screening of the synthesized com-
pounds was envisaged to provide the cytotoxicity infor-
mation.
Malaria is still one of the most deadly diseases
affecting more than 500 million people with over 1
million deaths per year.2 Combating malaria is an
important objective of WHO that established the Roll
Back Malaria as one of its highest priority programs.3
The 1,2,4,5-tetraoxacyclohexane (tetraoxane) moiety
became an increasingly interesting pharmacophore
since its antimalarial activity4 was found to be very
similar to that of 1,2,4-trioxanes such as naturally
occurring artemisinin,5 its semisynthetic derivatives,6
and related compounds.7 Therefore, substantial efforts
have been directed to the synthesis of 1,2,4,5-tetraox-
acycloalkanes and association of the antimalarial activ-
ity thereof, both in vitro and in vivo.8 Further develop-
ment of the tetraoxane carrier is additionally justified
by indications of reduced sensitivity of certain Plasmo-
dium falciparum strains to artemisinin.9
Ch em istr y
The design of an effective 1,2,4,5-tetraoxane-based
antimalarial drug meets several problems, among oth-
ers: (a) hydrophilic/lipophilic ratio of the carrier mol-
ecule; (b) stability under physiological conditions; (c)
cytotoxic/antimalarial activity ratio of the investigated
tetraoxane and of possible metabolites.
Few methods proved effective for the 1,2,4,5-tetraox-
acyclohexane (tetraoxane) ring synthesis. The hydrogen
peroxide approach (30%,4,8a,10 50%,8b and 70-90%11
H2O2) is the most widely used (along with various acid
catalysts), while the (TMSO)2/TMSOTf anhydrous pro-
cedure proved a good alternative to the above method.12
Recent methods8d,13 did not prove better (20-50%). At
present, all known procedures for tetraoxane synthesis
afford symmetrically 3,6-disubstituted 1,2,4,5-tetraox-
acyclohexane ring systems (only recently “mixed” tet-
raoxanes became available).14 Thus, when a single
enantiomer of a chiral ketone is used as starting
material, two diastereomers will be obtained.15 There-
fore, it is reasonable to anticipate different activity of
the diastereomers, providing additional information on
the structure-activity relationship (SAR). In this work
† Part of the projected dissertation of D.O., University of Belgrade.
* Corresponding author: Bogdan Sˇolaja, Faculty of Chemistry,
University of Belgrade, Studentski trg 16, P.O. Box 158, YU-11001
Belgrade, Yugoslavia. Phone: +381-11-63-86-06. Fax: +381-11-63-60-
61. E-mail: bsolaja@chem.bg.ac.yu.
#
Institute of Chemistry, Technology and Metallurgy.
3 Centro Internazionale di Servizi di Spettrometria di Massa del
CNR di Napoli.
National Cancer Research Institute.
§ Universite´ Catholique de Louvain.
| Walter Reed Army Institute of Research.
‡ University of Belgrade.
10.1021/jm000952f CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/09/2000

Cholic Acid Derivatives as Tetraoxane Carriers
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3275
Sch em e 1a
a
(a) Ac2O/TMSOTf/CH2Cl2, 86%; (b) K2CO3/MeOH, 96%; (c) J ones/acetone, 81%; (d) NaOH/CH2Cl2/MeOH, 93%; (e) benzene/SOCl2,
NH4Cl/Et3N/CH2Cl2, 61%; (f) benzene/SOCl2, n-PrNH2, 68%.
Sch em e 2a
a
(a) Toluene/H2O2/H2SO4, 0-5 °C; (b) CH2Cl2/(Me3Si)2O2/TMSOTf; (c) NaOH/CH2Cl2/MeOH.
we used the H2O2/H+ and (TMSO)2/TMSOTf procedures
and ketones 4, 6, and 7 (Scheme 2) as starting materials.
The required ketones were synthesized as shown in
Scheme 1. Methyl cholate was in three steps trans-
formed into ketone 4 of analytical purity (67% from 1).
Further steps include high-yield selective hydrolysis of
the methyl ester moiety (4 f 5, 93%) and preparation
of amides 6 (61%) and 7 (68%).
Tetraoxanes 8 and 9, possessing a methyl ester
functionality, were synthesized from ketone 5 using 32%
H2O2/H2SO4/H2O/EtOH/organic solvent mixtures at 0-5
°C (Scheme 2). The peroxyacetalization reaction was
examined in various organic solvents (and their mix-
tures) such as dichloromethane, dioxane, acetonitrile,
benzene, and toluene with the latter being the solvent
of choice. In other solvents a complex mixture of easily
decomposable products was obtained. Lower tempera-
tures (up to -78 °C), where applicable, did not improve
the tetraoxane yield, as well as the extension of the
reaction time to more than 2 h. Tetraoxanes with a
primary amide and n-propylamide moiety, 10/11 and
12/13, were synthesized using the (TMSO)2/TMSOTf
method12 from 6 and 7, respectively. It is interesting to
note that while the H2O2/H+ procedure worked well with
the C(24) ester functionality, it failed with primary and
secondary amides 6 and 7. On the contrary, the (TMSO)2/
TMSOTf method was sluggish with the C(24) esters.
The acids 14 and 15 were obtained by selective hydroly-
sis of corresponding methyl esters 8 and 9, respectively.
The tetraoxane structure of synthesized compounds
was deduced from their NMR spectra: each compound
exhibited the characteristic signal at ca. 108 ppm (OO-
C-OO moiety), Table 1. Liquid secondary ionization
(LSI) and electrospray ionization (ESI) mass spectra of
the above compounds yielded molecular ion peaks in all
cases. These experiments along with the LSIMS high-
resolution probe accurate mass measurements con-
firmed that tetraoxanes, and not hexaoxonanes (Table
1), were obtained.16
As expected, both diastereomeric tetraoxane series
were formed, and although the 1H and 13C NMR spectra
(CDCl3) of each pair are almost identical (at the level
of 200-MHz spectrometer), the clear distinction between
tetraoxanes within each pair can be made on the basis
of the appearance of the corresponding acetate methyl
group signals. In one series, the acetate methyls appear

3276 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Opsenica et al.
Ta ble 1. Physical and Selected Spectral Data of Tetraoxanes 8-15
NMR (CDCl3)
compd
mp (°C)
[R]2D0
1H acetate methyls
13C C(3) carbons
MS [method]a
8
9
251-252
167-170
211-217
196-199
240-243
171-174
228-232
199-202
+74.60
+50.75
+72.06
+44.80
+73.90
+41.66
+71.04
+49.07
2.10, 1.60b
2.12, 2.07, 1.64b
2.10
2.12, 2.07
2.10
2.12, 2.07
2.00c, 2.04c
2.12, 2.08
108.59, 108.91b, 108.67b
108.62, 108.77b
108.62
108.66
108.62
1063.8 (8) [M + Na]+, 1041.7 (10) [M + H]+ [ESI]
1063.5 (1) [M + Na]+, 1041.6 (12) [M + H]+ [ESI]
1011.6 (100) [M + H]+ [LSI]
10
11
12
13
14
15
1011.6 (80) [M + H]+ [LSI]
1095.7 (28) [M + H]+ [LSI]
108.66
1095.8 (100) [M + H]+ [LSI]
108.24c
1035.6 (43) [M + Na]+ [LSI]
108.68
1035.5 (100) [M + Na]+, 1013.5 (21) [M + H]+ [LSI]
a
b
See Experimental Section for conditions. Recorded in C6D6. c Recorded in DMSO-d6.
atomic parameters, and geometry are given as Support-
ing Information.
On the basis of the above data, we propose that
compounds 8, 10, 12 (and 14) belong to the cis series
(see Figure 1: cis-C(2),C(2a)), while 9, 11, 13 (and 15)
belong to the trans series. However, the conformation
of the presented compounds in solution remains un-
known at present.
Bioch em istr y
An tim a la r ia l Activity. Antimalarial activity of com-
pounds 8-15 against P. falciparum D6 and W2 clones
was investigated (Table 2) showing that esters 8 and 9,
as well as the corresponding acids 14 and 15, were
inactive against both clones. The amide moiety in
compounds 10, 11 and 12, 13, for the first time intro-
duced here as the auxiliary functional group in a
tetraoxane molecule, increases the antimalarial activity
of tetraoxanes 10-13, with primary amides being
somewhat more active against the P. falciparum chlo-
roquine-resistant W2 clone, while N-propylamides were
more active against the chloroquine-susceptible D6
clone. Most active was tetraoxane 12 with IC50 ) 9.29
nM against the D6 clone.
An tip r olifer a tive Activity. The antiproliferative
activity of all given compounds was tested against
human melanoma Fem-X and human cervix carcinoma
HeLa cell lines, and four compounds (8, 10, 13, and 14)
were chosen by NIH-NCI for in vitro screening (growth
inhibitory activities, GI) using a diverse panel of 60
human cancer cell lines.20 The tetraoxanes with the
N-propylamide moiety were also screened for cytotoxic
activity against normal human peripheral blood mono-
nuclear cells (PBMC) nonstimulated and stimulated
with phytohemaglutinine (PHA).
F igu r e 1. ORTEP plot of tetraoxane 8 (only position A for
the O-O bridge is shown). For more clarity, hydrogen atoms
are not represented. Cis substitution is apparent: C(2), C(2a)
same side of the tetraoxane ring.
as a broad singlet (at ca. 2.10 ppm), in the another as a
broad singlet (at ca. 2.12 ppm) followed by a smaller
broad singlet (at ca. 2.07 ppm) (Table 1), indicating that
the first series is C2 symmetrical. In addition, the
existence of two tetraoxane series is demonstrated by
respective [R]D values ([R]D(8, 10, 12, 14) ) 71-74; [R]D-
(9, 11, 13, 15) ) 42-50) and melting points (Table 1).
Tested tetraoxanes exhibit the antiproliferative activ-
ity at micromolar concentrations on various cell lines
(Table 2). IC50 values for Fem-X and HeLa cells were
determined after 72 h of continuous agent’s action by
MTT test, while GI50 values for 60 human cancer cell
lines were obtained applying a 48-h continuous drug
exposure protocol using SRB (sulforhodamine B) protein
assay.20
Methyl esters 8 and 9, as well as N-propylamide 13,
were the least active of all screened compounds with
IC50 not below ca. 20 µM on Fem-X and HeLa cells. GI50
MG_MID (meangraph midpoint) values derived for 8
and 13 are 81.3 and 57.5 µM, respectively, with 60
human cancer cell lines. The activity of tetraoxane 13
against renal cancers is quite interesting since it targets
only UO-31 (GI50 ) 0.10 µM) and 786-0 cell lines (GI50
) 4.36 µM). GI50 MG_MID for this compound is 57.5
The substitution pattern of the tetraoxane ring (cis
or trans) at C(3) and C(6) atoms (steroid numbering:
C(3) and C(3′), respectively), i.e., the structure of each
diastereomer, could not be determined on the basis of
NMR spectral data; however, the successful preparation
of the single crystal of tetraoxane 8 enabled us to solve
the problem by X-ray crystallographic structural analy-
sis. The tetraoxane 8 with C2 symmetry is shown in
Figure 1. Two positions (A and B) were found for the
O(36)-O(37) atoms (crystallographic numbering). The
six-membered 1,2,4,5-tetraoxane ring adopts a twisted
conformation, unlike the simple tetraoxanes.17 The
endocyclic torsion angles are: 82°, -44°, -40°, 82°,
-44°, -40°, ∆C2 ) 4° for position A (beginning with
C(3)-O(36)-O(37)-C(3*)) and -77°, 40°, 43°, -77°, 40°,
43°, ∆C2 ) 3° 18 for position B. The complete data,

Cholic Acid Derivatives as Tetraoxane Carriers
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3277
Ta ble 2. In Vitro Antimalarial and Antiproliferative Activities of Tetraoxanes 8-15
antiproliferative activity
IC50 (µM)a 72 h GI50 (µM)b 48 h
Fem-Xe
>29
P. falciparum IC50 (nM)a
D6c W2d
compd
HeLaf
cell line (MG_MID)
8
9
>96
>96
23.74
128.58
9.29
>96
>96
>29
20.5
3.7
6.0
33
CCRF-CEMg 14.8 (81.3)
19.5
3.1
6.2
23
94
4.2
7.6
10
11
12
13
14
15
18.79
HCC-2998h 0.97 (1.62)
59.35
60.43
30.12
20.08
166
5.2
9.2
UO-31i 0.10 (57.5)
DU-145 j 1.2 (2.0)
>99
>99
>99
7.3k
7.8
18
>99
artemisinin
8.6k
EC50(FCR-3)l
EC50(FCR-3)l
EC50(FM3A)m
EC50(FM3A)m
10
32
3.5
chloroquine
cis-platinum
a
b
IC, inhibition concentration. GI, growth inhibition. Presented are only the most sensitive cell lines to the corresponding drug out of
60. c P. falciparum African D6 clone. P. falciparum Indochina W2 clone. e Human melanoma. f Human cervix carcinoma. Leukemia.
d
g
Colon cancer. Renal cancer. Prostate cancer. Taken from ref 17. Chloroquine-sensitive FCR-3 strain.19
Mouse mammary tumor
h
i
j
k
l
m
FM3A.19
µM with GI50 for 35 cell lines exceeding a concentration
of 100 µM, meaning that the actual mean of GI50 is
>57.5 µM. In general, primary amides 10 and 11
exhibited more potent antiproliferative activity on Fem-X
and HeLa cells as compared to N-propylamide congeners
12 and 13. Moreover, the in vitro activity of tetraoxane
10 (as well as that of 14) is quite similar to that of cis-
platinum (Table 2). The mode of HeLa cells death
induced by tetraoxane 12 was analyzed using fluores-
cence microscopy, after cell staining with acridine
orange and ethidium bromide.21 Morphological exami-
nation of cells treated for 24 h with 2 × IC50 (66 µM) of
the above compound revealed that high concentration
of tetraoxane 12 induced fragmentation of nuclei into
many small clumps, typical for apoptosis.
Ta ble 3. Comparison of Antimalarial Activity and Cytotoxicity
of Tetraoxanes 12 and 13
antimalarial
activity
cytotoxicity IC50 (µM) 72 h
IC50 (nM)
selectivity
PHA-stimulated
PBMC (C)
compd D6 clone (A) PBMC (B)
B/A
C/A
12
13
9.29
20.08
>100
>320
96a
>10700 10333
>15900 12500
251a
a
Mean value of three people’s PHA-stimulated PBMC inhibition
is given in the text.
In Table 3 the inhibitory activity of 12 and 13 on the
P. falciparum D6 clone, as well as on PBMC and PHA-
stimulated PBMC, is compared. The high selectivity
values combined with pronounced antimalarial activity
of 12 indicate that tetraoxanes bearing the N-propyla-
mide moiety are good lead compounds for further
investigation.
Inspection of Tables 1 and 2 reveals also that the
antiproliferative activity of the cis series (cis: 10, 12,
14) is >2 times as great as that of the second diaster-
eomeric series (trans: 11, 13, 15). The antimalarial
activity of the amides follows the same trend, with the
exception of 12/13 ratio against the W2 clone.
Discu ssion
In this work steroidal tetraoxanes using a cholic acid
carrier were synthesized and their antimalarial and
antiproliferative activities evaluated. It was found that
an amide functionality renders enhanced antimalarial
activity in vitro as compared to methyl ester and
carboxylic acid moieties within the series of the conge-
ners. The antimalarial activity of tetraoxane 12 is high
(IC50 ) 9.29 nM), and 12 is almost as active as
artemisinin (Table 2). In addition, the N-propylamide
moiety significantly lowers the cytotoxicity within the
series.
Having in mind the relatively low cytotoxicity of
N-propylamides 12 and 13 on cancer cell lines and the
pronounced antimalarial activity of 12, they were
further screened for cytotoxic activity against normal
human peripheral blood mononuclear cells (PBMC)
nonstimulated and stimulated with phytohemaglutinine
(PHA) in 5 µg/mL concentration of nutrient medium.
PBMC were obtained from three healthy volunteers.
Determined IC50 values for nonstimulated PBMC were
>100 and >320 µM for tetraoxanes 12 and 13, respec-
tively. PHA-stimulated PBMC were somewhat less
resistant to cytotoxic action of the investigated com-
pounds with IC50 values of 98, 90, and 100 µM for
compound 12 (first, second, and third person’s PBMC)
and 270, 320, and 164 µM for compound 13. Being
affected by tetraoxanes 12 and 13 only at very high
concentrations, the PBMC can be considered as resis-
tant to their cytotoxic action.
Con clu sion
In this work the two diastereomeric series of steroidal
tetraoxanes with different bile acid side chain termini
were synthesized and characterized, and their biological
activity was evaluated. The antimalarial activity of bis-
(N-propylamide) 12 on the P. falciparum D6 clone is
very close to that of artemisinin, so considering its low
toxicity on PBMC (and on cancer cell lines tested) it
represents a good lead for further research in this area
(selectivity > 10 000). The antiproliferative activity of
some tetraoxanes is very close to that of cis-platinum,
while N-propylamide 13 targets almost only renal
cancer UO-31 out of the 60 cell lines tested, with GI50
) 0.10 µM, TGI ) 0.23 µM, and LC50 ) 0.54 µM.
Exp er im en ta l Section
Gen er a l. Melting points were determined on a Boetius
PMHK apparatus and were not corrected. Specific rotations
were determined on a Perkin-Elmer 141-MC at the given
temperatures. IR spectra were recorded on a Perkin-Elmer
spectrophotometer FT-IR 1725X. 1H and 13C NMR spectra were

3278 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Opsenica et al.
recorded on a Varian Gemini-200 spectrometer (at 200 and
50 MHz, respectively) in the indicated solvent using TMS as
internal standard. Chemical shifts are expressed in ppm (δ)
values and coupling constants (J ) in Hz.
3441m, 2965m, 2923m, 2875m, 1745s, 1713s, 1435m, 1382m,
1258s, 1238s, 1215m, 1072m cm-1 1H NMR (200 MHz,
.
CDCl3): 5.13 (bs, H-C(12)), 5.00 (d, J ) 2.8 Hz, H-C(7)), 3.66
(s, CH3O2C), 2.99 (dd, J ) 13.2, 13.2 Hz, HR-C(4)), 2.12 (s, CH3-
COO-), 2.07 (s, CH3COO-), 1.02 (s, H3C-C(10)), 0.82 (d, J )
6.2 Hz, H3C-C(20)), 0.77 (s, H3C-C(13)). 13C NMR (50 MHz,
CDCl3): 210.86, 173.20, 169.14, 168.86, 73.85, 69.20, 50.14,
45.97, 43.80, 43.15, 41.87, 40.75, 36.32, 35.21, 34.71, 33.17,
32.99, 29.44, 29.31, 28.38, 25.72, 24.40, 21.37, 20.22, 20.06,
19.90, 16.08, 10.82. MS (CI, isobutane, m/z): 506 (M+ + H),
445 (M+ - AcOH), 385 (M+ - 2× AcOH), 367 (M+ - 2× AcOH
- H2O).
Mass spectra were taken on the following spectrometers:
CI spectra were recorded on
a MS Finnigan-MAT 8230
spectrometer with double focusing reverse geometry, using
isobutane. LSI MS (FAB) spectra were recorded on a VG-
ZAB-T instrument equipped with a Cs ion gun. Accelerating
voltage was set to 8 kV using MNBA as matrix. Probe accurate
mass measurements were performed in the presence of PEG
internal callibrant at 5000 resolution. ESI MS spectra were
recorded on an Autospec TOF instrument in positive ion mode
using CH3CN:H2O (1:1) with 1% AcOH as carrying solvent
solution. The stock solutions of the samples were diluted in
order to obtain 10 pmol/µL solution using the carrying solvent.
The source temperature was 75 °C, and the cone voltage was
set to 40 V.
Thin-layer chromatography (TLC) was performed on pre-
coated Merck silica gel 60 F254 plates, using N,N-dimethyl-p-
phenylenediammonium dichloride peroxide reagent for per-
oxide moiety detection,22 and Lobar LichroPrep Si 60 (40-63
µm) columns coupled to a Waters RI 401 detector were used
for column chromatography.
3-Oxo-7r,12r-diacetoxy-5â-ch olan -24-oic Acid (5). Meth-
yl ester 4 (5.00 g, 9.9 mmol) was hydrolyzed at 80 °C with
NaOH (600 mg, 15 mmol) in i-PrOH/H2O mixture (80 mL, 3:1
v/v). Upon completion of the hydrolysis (TLC) the reaction
mixture was poured onto ice-cooled HCl acidified water. After
filtration and crystallization 4.53 g (93%) of acid 5 was
obtained. Mp ) 200-201.5 °C (colorless powder, ethanol) (lit.26b
mp ) 197 °C). [R]2D0 ) +56.70 (c ) 0.97, CHCl3). IR (KBr):
3500m, 2965m, 2878m, 1734s, 1713s, 1643w, 1437m, 1381s,
1254s, 1032m cm-1 1H NMR (200 MHz, CDCl3): 5.14 (bs,
.
H-C(12)), 5.01 (d, J ) 2.4 Hz, H-C(7)),), 3.00 (dd, J ) 15.0,
13.8 Hz, HR-C(4)), 2.12 (s, CH3COO-), 2.08 (s, CH3COO-),
1.02 (s, H3C-C(10)), 0.84 (d, J ) 6.2 Hz, H3C-C(20)), 0.77 (s,
H3C-C(13)). 13C NMR (50 MHz, CDCl3): 212.49, 179.56,
170.54, 170.27, 75.18, 70.56, 47.27, 45.03, 44.46, 43.19, 42.04,
37.64, 36.53, 36.02, 34.45, 34.31, 30.79, 30.72, 30.41, 29.70,
27.02, 25.73, 22.71, 21.56, 21.42, 21.27, 17.41, 12.18. MS (CI,
Met h yl 3r,7r,12r-Tr ia cet oxy-5â-ch ola n -24-oa t e (2).
Methyl cholate (1) (1.00 g, 2.37 mmol, mp ) 157-158 °C) was
dissolved in the previously prepared solution of Ac2O (1.00 mL)
and TMSOTf (26 µL, 0.14 mmol) at room temperature. After
stirring for 5 min, the reaction was quenched with saturated
NaHCO3 and worked-up as given in ref 23 to obtain 1.12 g
(86%) of 2 as light yellow oil. Crystallization from acetone/
hexane afforded pure 2. Mp ) 93-96 °C (colorless powder)
(lit.24 mp ) 90.5-91 °C). IR (film): 3021m, 2948s, 2871s,
isobutane, m/z): 491 (M+ + H), 431 (M+ - AcOH), 371 (M+
2× AcOH), 353 (M+ - 2× AcOH - H2O).
-
3-Oxo-7r,12r-diacetoxy-5â-ch olan -24-am ide (6). The acid
5 (3.00 g, 6.11 mmol) was dissolved in dry benzene (50 mL)
under inert atmosphere, cooled to 0 °C followed by addition of
SOCl2 (534 µL, 7.35 mmol). The reaction mixture was then
stirred at reflux for 3 h, cooled to room temperature, and
solvent evaporated under reduced pressure. Such obtained
crude acid chloride was dissolved in dry CH2Cl2 (25 mL), cooled
to 0 °C, and the suspension of NH4Cl (3.27 g, 61.1 mmol) and
Et3N (8.47 mL, 61.1 mmol) in dry CH2Cl2 (50 mL) was added.
The stirring was continued for 15 min at 0 °C and at room
temperature overnight. The reaction mixture was poured onto
water, extracted with CH2Cl2 (3× 30 mL), organic extracts
were washed with water and brine, and dried over anhydrous
Na2SO4. Crude product was purified by dry-flash chromatog-
raphy (SiO2) and after crystallization the pure amide 6 was
obtained (1.97 g, 61%). Mp ) 214 °C (colorless prisms, benzene/
hexane). [R]2D0 ) +57.59 (c ) 1.01, CHCl3). IR (KBr): 3426m,
2964m, 2878m, 1714s, 1688s, 1626m, 1380m, 1254s, 1032m
cm-1. 1H NMR (200 MHz, CDCl3): 5.46 (bs, NH2, exchangeable
with D2O), 5.14 (bs, H-C(12)), 5.00 (d, J ) 2.6 Hz, H-C(7)),
2.99 (dd, J ) 13.4, 13.4 Hz, HR-C(4)), 2.12 (s, CH3COO-), 2.07
(s, CH3COO-), 1.02 (s, H3C-C(10)), 0.84 (d, J ) 6.2 Hz, H3C-
C(20)), 0.77 (s, H3C-C(13)). 13C NMR (50 MHz, CDCl3):
212.18, 175.63, 170.47, 170.18, 75.22, 70.54, 47.47, 45.08,
44.52, 43.21, 42.098, 37.67, 36.58, 36.07, 34.67, 34.34, 32.65,
31.28, 30.85, 29.74, 27.13, 25.78, 22.74, 21.60, 21.45, 21.31,
17.56, 12.23. MS (CI, isobutane, m/z): 490 (M+ + H), 430 (M+
+ H - AcOH), 370 (M+ + H - 2× AcOH), 352 (M+ + H - 2×
AcOH - H2O). Anal. (C28H43NO6‚0.5H2O) C, H.
1734s, 1468m, 1438s, 1378s, 1245s, 1063s, 1022s, 756s cm-1
.
1H NMR (200 MHz, CDCl3): 5.09 (bs, H-C(12)), 4.91 (d, J )
2.6 Hz, H-C(7)), 4.65-4.50 (m, H-C(3)), 3.66 (s, CH3O2C(24)),
2.14 (s, CH3COO-), 2.09 (s, CH3COO-), 2.05 (s, CH3COO-),
0.92 (s, H3C-C(10)), 0.81 (d, J ) 6.2 Hz, H3C-C(20)), 0.73 (s,
H3C-C(13)). 13C NMR (50 MHz, CDCl3): 174.6, 170.5, 170.4,
75.36, 74.05, 70.6, 51.5, 47.3, 45.0, 43.4, 40.9, 37.7, 34.6, 34.3,
31.2, 30.8, 30.7, 28.8, 27.1, 26.8, 25.5, 22.7, 22.5, 21.6, 21.5,
17.4, 12.1. MS (CI, isobutane, m/z): 549 (M+ + H), 489 (M+
AcOH), 429 (M+ - 2× AcOH), 369 (M+ - 3× AcOH).
-
Met h yl 3r-H yd r oxy-7r,12r-d ia cet oxy-5â-ch ola n -24-
oa te (3). Triacetate 2 (5.30 g, 9.66 mmol) was dissolved in
dry methanol (80 mL), 2.40 g (17.4 mmol) of anhydrous K2-
CO3 was added and the suspension was stirred at room
temperature for 2 h. The reaction was quenched by addition
of acetic acid (3 mL), after CO2 was liberated resulting solution
was evaporated under reduced pressure, and the residue was
transferred as EtOAc suspension to the separatory funnel. The
organic layer was well washed with brine and dried over
anhydrous Na2SO4. Crystallization of the crude product from
EtOAc/hexane mixture afforded methyl 3R-hydroxy-7R,12R-
o
triacetoxy-5â-cholan-24-oate (3; 4.72 g, 96%). Mp ) 59-62 C
(colorless plates) (lit.25 mp ) 57-59 oC). IR (KBr): 3446m,
2953s, 2870s, 1735s, 1654m, 1467m, 1379s, 1245s, 1101s,
1075s, 1022s cm-1 1H NMR (200 MHz, CDCl3): 5.08 (bs,
.
H-C(12)), 4.90 (d, J ) 2.6 Hz, H-C(7)), 3.66 (s, CH3O2C(24)),
3.60-3.40 (m, H-C(3)), 2.13 (s, CH3COO-), 2.09 (s, CH3COO-
), 0.91 (s, H3C-C(10)), 0.81 (d, J ) 6.0 Hz, H3C-C(20)), 0.73
(s, H3C-C(13)). 13C NMR (50 MHz, CDCl3): 173.2, 169.4,
73.97, 69.9, 69.4, 50.0, 47.7, 43.5, 41.8, 39.5, 36.99, 36.2, 33.4,
33.0, 32.8, 29.8, 29.3, 29.2, 28.8, 27.4, 25.6, 23.99, 21.2, 21.06,
N -(n -P r op yl)-3-oxo-7r,12r-d ia ce t oxy-5â-ch ola n -24-
a m id e (7). Starting with acid 5 (2.95 g, 6.01 mmol) the same
procedure was applied as for 6, only replacing the NH4Cl/Et3N
mixture by 2 equiv of n-PrNH2. After crystallization of the
crude product from benzene/hexane mixture, N-propylamide
7 was obtained (2.17 g, 68%). Mp ) 216 °C (colorless needles,
benzene/hexane). [R]2D0 ) +50.10 (c ) 1.00, CHCl3). IR (KBr):
3407m, 2954m, 2935m, 2871m, 1735s, 1718s, 1664s, 1534m,
20.12, 19.9, 15.9, 10.6. MS (CI, isobutane, m/z): 507 (M+
+
H), 447 (M+ + H - AcOH), 387 (M+ + H - 2× AcOH), 369
(M+ + H - 2× AcOH - H2O).
Meth yl 3-Oxo-7r,12r-d ia cetoxy-5â-ch ola n -24-oa te (4).
3-Hydroxy compound 3 (2.00 g, 3.95 mmol) was oxidized by
J ones reagent under standard reaction conditions to afford
after repeated crystallization of the crude product ketone 4
(1.61 g, 81%). Mp ) 196-198 °C (colorless prisms; benzene/
hexane). [R]2D0 ) +57.08 (c ) 0.97, CHCl3) (lit.25 mp ) 190-
191 °C; lit.25 [R]D20 ) +61.60 (c ) 2.0, CHCl3)). IR (KBr):
1376m, 1258s, 1244s, 1028m cm-1 1H NMR (200 MHz,
.
CDCl3): 5.59-5.43 (m, H-N, exchangeable with D2O), 5.13
(bs, H-C(12)), 5.00 (d, J ) 2.8 Hz, H-C(7)), 3.30-3.17 (m,
CH3CH2CH2N), 2.99 (dd, J ) 13.4, 13.4 Hz, HR-C(4)), 2.11 (s,
CH3COO-), 2.07 (s, CH3COO-), 1.57-1.43 (m, CH3CH2CH2N),
1.02 (s, H3C-C(10)), 0.92 (t, J ) 7.4 Hz, CH3CH2CH2N), 0.83

Cholic Acid Derivatives as Tetraoxane Carriers
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3279
(d, J ) 6.2 Hz, H3C-C(20)), 0.77 (s, H3C-C(13)). 13C NMR
(50 MHz, CDCl3): 212.18, 175.63, 170.47, 170.18, 75.22, 70.54,
47.47, 45.08, 44.52, 43.21, 42.10, 37.67, 36.58, 36.07, 34.67,
34.34, 32.65, 31.28, 30.85, 29.74, 27.13, 25.785, 22.74, 21.60,
21.45, 21.31, 17.56, 12.23. MS (CI, isobutane, m/z): 532 (M+
+ H), 472 (M+ + H - AcOH), 412 (M+ + H - 2× AcOH). Anal.
(C31H49NO6) C, H.
methods27 and refined by full-matrix least-squares on F2.28
Final R values: R1 ) 0.099 for 3193 reflections with I > 2σ(I),
R1 ) 0.101 for all data, wR2 ) 0.295. The oxygen atoms of the
tetraoxane O(36) and O(37) (Figure 1, crystallogragraphic
numbering) occupy two positions (A and B) with site occupa-
tion factors of 0.52 and 0.48, respectively. In the final Fourier
difference synthesis, four peaks of electron density ranging
from 1.2 to 0.8 e Å-3 could not be interpreted; this explains
probably the relatively high R indices. The cocrystallized
aceton molecule is located on the 2-fold axis.
Bis(m eth yl 3-dioxy-7r,12r-diacetoxy-5â-ch olan -24-oate)
(8 a n d 9). A solution of methyl ester 4 (400 mg, 0.79 mmol)
in toluene (8.3 mL) was added to previously cooled (0 °C)
solution of EtOH (1.20 mL), H2O (1.12 mL) and H2SO4 (2.16
mL). After 15 min, 32% H2O2 (200 µL) was added and intensive
stirring was continued next 2 h at 0 °C, when the reaction
mixture was diluted with H2O (20 mL) and toluene (30 mL).
The organic layer was washed with water (2 × 10 mL),
saturated NaHCO3 (2 × 10 mL), brine, and dried over
anhydrous Na2SO4. Column chromatography (Lobar B, Li-
chroPrep Si 60, eluent: heptane/EtOAc (7:3)) of the obtained
complex mixture afforded two main fractions, which upon
crystallization afforded 8 (104 mg, 25%) and 9 (116 mg, 28%).
8: mp ) 251-252 °C (colorless prisms, hexane/acetone).
[R]2D0 ) +74.60 (c ) 1.10, CHCl3). IR (KBr): 2995m, 1737s,
Bis(3-dioxy-7r,12r-diacetoxy-5â-ch olan -24-oic acid) (14).
Methyl ester 8 (250 mg, 0.24 mmol) was dissolved in CH2Cl2/
CH3OH (1:1) mixture (100 mL), 1.25 M NaOH (29.95 mL)
solution was added at room temperature and the clear reaction
mixture was stirred for 3 days. (1.25 M solution of NaOH was
prepared by dissolving 2.50 g NaOH in 50 mL of MeOH:H2O
) 95:5 (v/v) mixture.) The reaction was quenched by addition
of glacial AcOH (until pH 5), CH2Cl2 (30 mL) and water (50
mL) were added, and separated water layer was extracted with
CH2Cl2 (5× 30 mL). Combined organic layers were washed
with water and brine, dried over anhydrous Na2SO4, and
evaporated to dryness. Crystallization from acetone/hexane
gave 192 mg (79%) of 14 as colorless powder. Mp ) 228-232
°C. [R]2D0 ) +71.04 (c ) 1.10, DMSO). IR (KBr): 3450s, 2953s,
1737s, 1441w, 1380s, 1253s, 1126w, 1081m cm-1. 1H NMR (200
MHz, DMSO-d6): 11.94 (bs, 2 × HO2C(24) exchangeable with
D2O), 4.98 (bs, 2 × H-C(12)), 4.82 (bs, 2 × H-C(7)), 2.04 (s,
ca. 2 × CH3COO-), 2.00 (s, ca. 2 × CH3COO-), 0.91 (s, 2 ×
H3C-C(10)), 0.74 (d, J ) 6.0 Hz, 2 × H3C-C(20)), 0.70 (s, 2 ×
H3C-C(13)). 13C NMR (50 MHz, DMSO-d6): 175.04, 169.97,
169.84, 108.24, 74.70, 70.28, 47.17, 44.77, 43.19, 36.88, 34.35,
30.84, 30.59, 28.00, 26.83, 25.37, 22.395, 22.12, 21.43, 21.08,
17.38, 12.09. MS (LSI, m/z): 1035.6 ([M + Na]+, 43), 1012.6
(4), 993.6 (23), 969.6 (19), 951.6 (34), 891.5 (100), 849.5 (29),
794.0 (26), 780.0 (45), 766.1 (27), 749.1 (29), 735.0 (66%), 719.0
(34), 691.0 (36), 674.0 (47), 658.0 (42), 643.0 (53), 631.0 (58).
Anal. (C58H84O16‚2H2O) C, H.
1638w, 1440m, 1378m, 1250s, 1027m cm-1 1H NMR (200
.
MHz, CDCl3): 5.09 (bs, 2 × H-C(12)), 4.92 (bs, 2 × H-C(7),
3.66 (s, 2 × CH3O2C(24)), 2.10 (bs, 4 × CH3COO-), 0.94 (s, 2
× H3C-C(10)), 0.81 (d, J ) 5.8 Hz, 2 × H3C-C(20)), 0.73 (s,
2 × H3C-C(13)). 13C NMR (50 MHz, CDCl3): 174.48, 170.45,
108.59, 75.16, 70.52, 51.42, 47.196, 44.92, 43.19, 37.55, 34.52,
34.47, 32.05, 30.72, 30.61, 28.32, 27.04, 25.57, 22.67, 22.03,
21.54, 21.23, 17.37, 12.09. 1H NMR (200 MHz, C6D6): 5.16 (bs,
2 × H-C(12)), 4.94 (bs, 2 × H-C(7)), 3.40 (s, 2 × CH3O2C-
(24)), 1.60 (bs, 2 × CH3COO-), 0.80 (d, J ) 6.0 Hz, 2 × H3C-
C(20)), 0.60 (s, 2 × H3C-C(10)), 0.44 (s, 2 × H3C-C(13)). 13C
NMR (50 MHz, C6D6): 173.64, 169.53, 108.91, 108.67, 75.00,
70.43, 50.93, 47.65, 45.23, 43.81, 37.71, 34.80, 34.65, 31.01,
30.80, 30.6, 28.72, 27.30, 25.99, 22.95, 22.02, 22.09, 20.56,
17.56, 12.17. MS (ESI, m/z): 1063.8 ([M + Na]+, 8), 1041.7
([M + H]+, 10), 981.6 (4), 921.7 (14), 597.0 (13), 579.0 (20),
537.4 (61), 477.3 (50), 417.3 (18), 238.0 (60), 197.0 (100), 178.2
(54), 149.0 (35). Anal. (C58H88O16) C, H. 9: mp ) 167-170 °C
(colorless prisms, hexane/acetone). [R]2D0 ) +50.75 (c ) 1.10,
CHCl3). IR (KBr): 2960m, 1738s, 1439m, 1378m, 1238s, 1026m
Bis(3-dioxy-7r,12r-diacetoxy-5â-ch olan -24-oic acid) (15).
Starting with methyl ester 9 (130 mg, 0.12 mmol), the same
procedure was repeated as with 8. After crystallization from
CH2Cl2/i-Pr2O the acid 15 was obtained as colorless powder
(91 mg, 72%). Mp ) 199-202 °C. [R]2D0 ) +49.07 (c ) 1.02,
CHCl3). IR (KBr): 3473m, 3456m, 2953s, 1737s, 1441m, 1380s,
cm-1
.
1H NMR (200 MHz, CDCl3): 5.09 (bs, 2 × H-C(12)),
1250s, 1125w, 1081w, 1027m cm-1 1H NMR (200 MHz,
.
4.92 (bs, 2 × H-C(7)), 3.66 (s, 2 × CH3O2C(24)), 2.12 (bs, ca.
2 × CH3COO-), 2.07 (bs, ca. 2 × CH3COO-), 0.94 (s, 2 ×
H3C-C(10)), 0.81 (d, J ) 5.8 Hz, 2 × H3C-C(20)), 0.73 (s, 2 ×
H3C-C(13)). 13C NMR (50 MHz, CDCl3): 174.51, 170.47,
108.62, 75.23, 70.59, 51.47, 47.27, 44.98, 43.24, 37.56, 34.58,
34.52, 30.78, 30.66, 28.33, 27.08, 25.62, 22.71, 22.02, 21.47,
CDCl3): 5.09 (bs, 2 × H-C(12)), 4.93 (bs, 2 × H-C(7)), 2.12
(bs, ca. 2 × CH3COO-), 2.08 (bs, ca. 2 × CH3COO-), 0.94 (s,
2 × H3C-C(10)), 0.82 (d, J ) 5.6 Hz, 2 × H3C-C(20)), 0.73 (s,
2 × H3C-C(13)). 13C NMR (50 MHz, CDCl3): 179.48, 170.56,
170.38, 108.68, 75.29, 70.66, 47.29, 45.03, 43.28, 37.64, 34.62,
34.51, 30.68, 30.46, 28.39, 27.11, 25.66, 22.74, 22.05, 21.52,
21.36, 17.45, 12.18. MS (LSI, m/z): 1035.6 ([M + Na]+, 100),
1013.5 ([M + H]+, 21), 1011.4 (17), 993.5 (27), 951.4 (41), 909.4
(11), 893.4 (51), 849.5 (12), 833.4 (17), 779.9 (14), 734.9 (17),
1
21.31, 17.41, 12.13. H NMR (200 MHz, C6D6): 5.15 (bs, 2 ×
H-C(12)), 4.95 (bs, 2 × H-C(7)), 3.38 (s, 2 × CH3O2C(24)),
1.64 (bs, 4 × CH3COO-), 0.80 (d, J ) 5.2 Hz, 2 × H3C-C(20)),
0.66 (s, 2 × H3C-C(10)), 0.45 (s, 2 × H3C-C(13)). 13C NMR
(50 MHz, C6D6): 173.64, 169.55, 108.77, 75.02, 70.48, 50.93,
47.67, 45.25, 43.89, 38.08, 37.71, 34.78, 34.67, 32.56, 31.03,
30.80, 30.60, 28.85, 27.28, 26.02, 25.06, 22.93, 22.06, 20.82,
20.53, 17.54, 12.19. MS (ESI, m/z): 1063.5 ([M + Na]+, 1),
1041.7 ([M + H]+, 12), 981.6 (1), 921.5 (6), 537.3 (51), 477.3
(75), 417.3 (47), 385.3 (45), 351.2 (17), 307.0 (100). Anal.
(C58H88O16) C, H.
699.5 (15), 673.9 (13), 644.9 (16), 628.9 (15). Anal. (C58H84O16
C, H.
)
Bis(3-d ioxy-7r,12r-d ia cetoxy-5â-ch ola n -24-a m id e) (10
a n d 11). Using procedure as given in ref 12, 6 (500 mg, 1.02
mmol) was treated with TMSOTf/TMS2O2 solution in CH3CN.
The reaction mixture was poured onto well-stirred benzene/
NaHCO3/ice/water mixture, extracted with benzene, washed
with brine and dried over anhydrous Na2SO4. Column chro-
matography (Lobar B, LichroPrep Si 60, eluent: EtOAc/THF
(7:3)) of the obtained complex mixture afforded two main
fractions, which upon crystallization afforded 10 (133 mg, 26%)
and 11 (124 mg, 24%). 10: mp ) 211-217 °C (colorless powder,
X-r a y An a lysis of 8. A first data set was collected at room
temperature but the structure could not be refined properly
because of disorder and high thermal agitation in the lateral
chain and for the atoms in the vicinity of C(3). With the hope
to solve these disorder problems, it was decided to collect a
new data set at low temperature (100 K).
The crystal (0.3 × 0.3 × 0.2 mm) was mounted in inert oil
and transferred to the cold gas stream of a MAR345 image
plate equipped with Mo KR graphite monochromatized radia-
tion (λ ) 0.71069 Å). Crystal data for C58H88O16‚C3H6O are as
follows: Mr ) 1097.35, orthorhombic, space group P21212; a
) 23.525(8), b ) 8.606(3), c )15.982(5) Å; V ) 3236(2) Å3, Z )
2. A total of 36415 reflections were measured; 3356 were
independent (Rint ) 0.051). The structure was solved by direct
CH2Cl2/i-Pr2O). [R]20 ) +72.06 (c ) 1.30, CHCl3). IR (KBr):
D
3452s, 2955s, 2873m, 1718s, 1672s, 1443m, 1379s, 1256s,
1166w, 1126m, 1104m, 1080w, 1029w, 967w cm-1 1H NMR
.
(200 MHz, CDCl3): 5.45 (bs, 2 × NH2), 5.09 (bs, 2 × H-C(12)),
4.92 (bs, 2 × H-C(7)), 2.10 (bs, 4 × CH3COO-), 0.94 (s, 2 ×
H3C-C(10)), 0.83 (d, J ) 5.8 Hz, 2 × H3C-C(20)), 0.73 (s, 2 ×
H3C-C(13)). 13C NMR (50 MHz, CDCl3): 175.70, 170.54,
108.62, 75.23, 70.59, 47.41, 45.01, 43.24, 37.62, 34.60, 32.65,
31.30, 30.59, 28.39, 27.15, 25.64, 22.74, 22.09, 21.62, 21.32,

3280 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Opsenica et al.
17.54, 12.20. MS (LSI, m/z): 1011.6 ([M + H]+, 100), 967.7
(17), 909.6 (16), 891.6 (29), 851.6 (15), 793.6 (17), 766.4 (20),
735.5 (20), 677.5 (24), 647.5 (15), 613.2 (95), 577.4 (14), 561.4
(20). Anal. (C56H86N2O14‚3H2O) C, H. 11: mp ) 196-199 °C
automatically by a Biomek 1000 or 2000 liquid handling
system into 96-well microtiter plates. The diluted drugs were
then transferred (25 µl) to test plates, 200 µl of parasitized
erythrocytes (0.2% parasitemia and 1% hematocrit) was added,
and the whole was incubated at 37 °C in a controlled environ-
ment of 5% CO2, 5% O2 and 90% N2. After 42 h, 25 µLof [3H]-
hypoxanthine was added and the plates were incubated for
an additional 24 h. At the end of the 66-h incubation period,
the plates were frozen at -70 °C to lyse the red cells and later
thawed and harvested onto glass fiber filter mats by using a
96-well cell harvester. The filter mats were then counted in a
scintillation counter and the data downloaded with the custom,
automated analysis software developed at WRAIR. For each
drug, the concentration-response profile was determined and
50% inhibitory concentrations (IC50) were determined by using
a nonlinear, logistic dose-response analysis program.
An tip r olifer a tive Activity. Ma ter ia ls a n d m eth od s:
Stock solutions of investigated compounds were prepared in
DMSO at a concentration of 8 mM and afterward diluted by
nutritient medium (RPMI-1640 medium supplemented with
L-glutamine (3 mmol/L), streptomycin (100 µg/mL), and peni-
cillin (100 IU/mL), 10% heat-inactivated FBS and 25 mM
Hepes, adjusted to pH 7.2 by bicarbonate solution) to various
final concentrations. The 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT; Sigma Chemicals) was
dissolved, 5 mg/mL in phosphate buffer saline, pH 7.2, and
filtered through a Millipore filter, 0.22 µm, before use. RPMI-
1640 cell culture medium and FBS were products of Sigma
Chemicals.
(colorless powder, CH2Cl2/i-Pr2O). [R]20 ) +44.80 (c ) 1.20,
D
CHCl3). IR (KBr): 3452s, 2955s, 2877m, 1738s, 1670s, 1441m,
1379s, 1255s, 1166w, 1125m, 1081m, 1027w, 966w cm-1 1H
.
NMR (200 MHz, CDCl3): 5.42 (bs, 2 × NH2), 5.10 (bs, 2×
H-C(12)), 4.92 (bs, 2 × H-C(7)), 2.12 (bs, ca. 2 × CH3COO-
), 2.07 (bs, ca. 2× CH3COO-), 0.94 (s, 2 × H3C-C(10)), 0.83
(d, J ) 6.0 Hz, 2 × H3C-C(20)), 0.73 (s, 2 × H3C-C(13)). 13C
NMR (50 MHz, CDCl3): 175.64, 170.51, 170.31, 108.66, 75.27,
70.63, 47.43, 45.03, 43.26, 37.60, 34.615, 34.22, 32.67, 32.03,
31.30, 30.50, 28.35, 27.15, 25.66, 22.73, 22.03, 21.51, 21.36,
17.54, 12.20. MS (LSI, m/z): 1011.6 ([M + H]+, 80), 967.6 (6),
919.3 (10), 851.6 (6), 766.2 (14), 735.5 (10), 677.5 (11), 613.2
(100), 581.2 (9), 566.2 (11). Anal. (C56H86N2O14) C, H.
Bis(N-(n -p r op yl)-3-d ioxy-7r,12r-d ia cetoxy-5â-ch ola n -
24-a m id e) (12 a n d 13). Using the same procedure as above,
7 (532 mg, 1.00 mmol) was transformed into 12 (74 mg, 14%)
and 13 (62 mg, 11%) after chromatography of the crude product
(Lobar B, LichroPrep Si 60, eluent: EtOAc). 12: mp ) 240-
243 °C (colorless powder, CH2Cl2/i-Pr2O). [R]20 ) +73.90 (c )
D
1.40, CHCl3). IR (KBr): 3422m, 2960s, 2874m, 1738s, 1650s,
1551m, 1441m, 1378s, 1250s, 1027m cm-1. 1H NMR (200 MHz,
CDCl3): 5.59-5.43 (m, 2 × H-N), 5.09 (bs, 2 × H-C(12)), 4.93
(bs, 2 × H-C(7)), 3.28-3.12 (m, 2 × CH3CH2CH2N), 2.10 (bs,
4 × CH3COO-), 1.60-1.40 (m, 2 × CH3CH2CH2N), 0.96-0.87
(m, 2 × H3C-C(10), 2 × CH3CH2CH2N), 0.82 (d, J ) 5.8 Hz,
H3C-C(20)), 0.73 (s, H3C-C(13)). 13C NMR (50 MHz, CDCl3):
173.19, 170.51, 108.62, 75.24, 70.57, 47.44, 44.98, 43.21, 41.11,
37.59, 34.67, 34.56, 33.53, 31.52, 30.59, 28.35, 27.11, 25.60,
22.81, 22.04, 21.55, 21.28, 17.52, 12.15, 11.27. MS (LSI, m/z):
1095.7 ([M + H]+, 28), 1035.7 (3), 613.1 (3), 532.4 (5), 504.4
(4) 460.2 (14), 412.3 (10), 307.0 (100). Anal. (C62H98N2O14) C,
H. 13: mp ) 171-174 °C (colorless powder, CH2Cl2/i-Pr2O).
[R]2D0 ) +41.66 (c ) 1.40, CHCl3). IR (KBr): 3951m, 3424m,
2960m, 2875m, 1738s, 1651m, 1547m, 1441m, 1379s, 1250s,
Cell cu ltu r e: Human malignant melanoma Fem-X cells
and human cervix carcinoma HeLa cells were maintained as
a monolayer culture in the same nutrient medium. The cells
were grown at 37 °C in 5% CO2 and humidified air atmosphere
by twice weekly subculture.
Tr ea tm en t of F em -X a n d HeLa cells: Target cells were
seeded, in triplicate (2000 cells/well), into 96-well microtiter
flat bottomed plates and 20 h later, five different concentra-
tions of investigated compounds were added to the wells to
various final concentrations, except to the control wells where
a nutrient medium with correspondent concentration of DMSO
only was added to the cells. All analyses were done in
triplicate. Nutrient medium with corresponding concentrations
of compounds, but void of cells was used as blank, in triplicate
too.
1165w, 1125w, 1026m, 966w cm-1 1H NMR (200 MHz,
.
CDCl3): 5.38-5.49 (m, 2 × H-N), 5.09 (bs, 2 × H-C(12)), 4.92
(bs, 2 × H-C(7)), 3.28-3.13 (m, 2 × CH3CH2CH2N), 2.12 (bs,
ca. 2 × CH3COO-), 2.07 (bs, ca. 2 × CH3COO-), 1.60-1.40
(m, 2 × CH3CH2CH2N), 0.94 (s, 2 × H3C-C(10)), 0.96-0.86
(m, 2 × H3C-C(10), 2 × CH3CH2CH2N), 0.82 (d, J ) 6.0 Hz,
2 × H3C-C(10)), 0.73 (s, 2 × H3C-C(13)). 13C NMR (50 MHz,
CDCl3): 173.18, 170.54, 108.66, 75.29, 70.63, 47.49, 45.01,
43.26, 41.13, 37.60, 34.71, 34.60, 33.60, 31.56, 28.35, 27.15,
25.66, 22.85, 22.73, 22.03, 21.525, 21.38, 17.54, 12.18, 11.31.
MS (LSI, m/z): 1095.8 ([M + H]+, 100), 1035.8 (5), 967.7 (2),
909.6 (2), 851.7 (2), 793.6 (3), 766.2 (4), 677.5 (3), 613.2 (16).
Anal. (C62H98N2O14‚2H2O) C, H.
An tim a la r ia l Activity. The in vitro antimalarial drug
susceptibility screen was a modification of the procedures first
published by Desjardins et al.,29 with modifications developed
by Milhous et al.30 In brief, the assay relies on the incorpora-
tion of radiolabeled hypoxanthine by the parasites and inhibi-
tion of isotope incorporation is attributed to activity of known
or candidate antimalarial drugs. For each assay, proven
antimalarials were used as controls. The incubation period was
66 h and the starting parasitemia was 0.2% with a 1%
hematocrit. The medium used was RPMI-1640 culture medium
with no folate or p-aminobenzoic acid (PABA) and 10% normal
heat-inactivated human plasma. For quantitative in vitro drug
susceptibility testing, two well-characterized P. falciparum
malaria clones were normally used, W2 and D6.31 W2 is a clone
of the Indochina I isolate and is resistant to chloroquine and
pyrimethamine but susceptible to mefloquine. D6 is a clone
from the Sierra I/UNC isolates and is susceptible to chloro-
quine and pyrimethamine but has reduced susceptibilities to
mefloquine and halofantrine
Deter m in a tion of HeLa a n d F em -X cell su r viva l: Cell
survival was determined as that reported earlier32 by MTT
test 72 h after the drug addition. In brief, 20 µL of MTT
solution (5 mg/mL PBS) was added to each well. Samples were
incubated for a further 4 h at 37 °C in 5% CO2 and humidified
air atmosphere. Then, 100 µL of 10% SDS in 0.01 M HCl was
added to the wells. Optical density (OD) at 570 nm was read
the next day. To obtain cell survival (%), optical density at
570 nm of a sample with cells grown in the presence of various
concentration of investigated agent (OD) was divided with
control optical density ODc (the OD of cells grown only in
nutritient medium) × 100. (It was implied that the OD of the
blank was always subtracted from the OD of a corresponding
sample with target cells.) Concentration IC50 was defined as
the concentration of a drug needed to inhibit cell survival by
50%, compared with vehicle-treated control. The NIH-NCI
screening results are given in growth inhibitory concentrations
(GI). The GI50 is the interpolated value representing the
concentration at which the percentage growth is +50.
P r ep a r a tion of p er ip h er a l blood m on on u clea r cells
(P BMC): PBMC were separated from whole-heparinized blood
of three healthy volunteers by Lymphoprep (Oslo, Norway)
gradient centrifugation. Interface cells, washed three times
with nutrient medium, were counted and resuspended in the
same medium. Then, 100 000 PBMC were seeded in 100 µL
of nutrient medium with or without PHA, and 2 h later 50 µL
of investigated tetraoxanes in various dilutions was put into
the wells in triplicate; in the control, sample nutrient medium
was added only. Final concentration of PHA was 5 µg/mL of
Drugs were dissolved directly in dimethyl sulfoxide (DMSO)
and diluted 400-fold with complete culture media. The com-
pounds were then diluted 2-fold, 11 times, to give a concentra-
tion range of 1048-fold. These dilutions were performed

Cholic Acid Derivatives as Tetraoxane Carriers
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3281
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nutrient medium. Determination of PBMC survival and IC50
was done in the same way as reported for HeLa and Fem-X
cells.
Ack n ow led gm en t. We thank the NIH-NCI’s De-
velopmental and Therapeutics program for evaluation
of our tetraoxanes.
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Su p p or tin g In for m a tion Ava ila ble: Complete crystal
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